To determine whether p53 manipulation may alter the metabolic sensitivities of EAC cells, baseline assessment of EAC cell sensitivity to metabolic inhibitors and to specific p53 drugs is necessary. Metformin blocks complex I of the ETC thereby preventing mitochondrial respiration and 2-deoxyglucose (2-DG) acts as a glucose mimic and blocks glycolysis. Given that both of these drugs are easily obtainable and effects are well known in the literature, we chose these drugs to establish baseline data for the effectiveness of metabolic inhibition in EAC cells. Multiple p53 mutant inhibitors and p53 wild-type activators are available. RETRA targets the binding of p53 mutant protein to its associated protein, p73, thereby restoring the function of p73. Pifithrin inhibits p53 activity. Prima1 targets p53 mutations and reactivates wild-type activity. NSC59984 (NSC) is a compound in the National Cancer Institute chemical diversity library that has recently been shown in colorectal cancer to restore p53 wild-type function by activating p73 and simultaneously degrades mutant p53. All of these drugs have potentially valuable effects, however, NSC and Prima1 reactivate wild-type p53 and inhibit mutant p53 which would potentially restore the advantageous aspects of p53 while preventing its gain-of-function activities. Additionally, NSC was synthesized within the NCI which simplifies using this drug in clinical trials. Accomplishments: We established a baseline by first investigating inhibition of cell lines with metabolic inhibitors. All cells responded with a dose dependent reduction in proliferation to metformin and 2-DG. When these inhibitors were used in combination, the reduction in proliferation varied across the panel of cells, however, the effects were uniformly more robust. Additionally, p53 wild-type Barrett's line CP-A was less sensitive to metabolic inhibitors alone than EAC cells. Unfortunately, we have no additional cell lines with wild-type p53 (Specific Research Aim 2). To determine the effects of both 2-DG and metformin on OXPHOS or glycolysis, OCR and ECAR were measured with the Seahorse Bioscience XF96 Extracellular Flux Analyzer. As expected, these results consistently reveal that metformin blocked maximal mitochondrial respiratory capacity whereas 2-DG reduced glycolysis with minimal impact on the mitochondrial respiration. These measurements have set up the basis for further evaluation of the effects of p53 mutant inhibitors on metabolism. Next, we investigated inhibition with p53 targeted inhibitors alone and in combination with metabolic inhibitors and standard chemotherapy. Based on the specific mutations defined in Specific Research Aim 2, CP-A, NCI-SB-Esc3, and Eso26 were selected for additional studies. Multiple compounds were initially evaluated, but NSC was chosen for additional treatment and mechanistic experiments. Although we are most interested in the combination of p53 mutant inhibitors with metabolic inhibition, the standard, approved treatments in esophageal cancer includes platinum-based therapy, therefore, we used combination with cisplatin (Cis) as well as metformin (Met). These results reveal that as single agents, both NSC and metformin significantly reduces proliferation of EAC cells. The reduction in proliferation was greater with both in combination than as signal agents. Interestingly, the combination of NSC and cisplatin appeared more robust than metformin and cisplatin. A synergistic reduction in proliferation was noted p53-mutant cells compared to the p53 wild-type cells. Similar results were obtained when apoptosis was measured with cisplatin and NSC (Figure 21). These results suggest that the mutational status is relevant for treatment approach. At this point, these experiments need to be confirmed with a greater number of cell lines and the mechanisms of action need to be explored further. To test whether NSC alters mitochondrial function through known proteins that link p53 to the mitochondria, expression analysis, localization studies, and immunoprecipitation experiments where performed in cells treated with NSC versus controls. In the p53 mutant cell lines, Esc3 and Eso26, NSC dose-dependent increases occurred in the proteins p73, TIGAR, SCO2, mitochondrial fusion genes (Mfn1, Mfn2, Opa1) as noted by qRT-PCR and immunoblot. Additionally, mitochondrial elongation was seen by confocal imaging. We evaluated whether p53 directly interacted with SCO2 and TIGAR once treated with NSC. Both SCO2 and TIGAR appeared to directly bind to p53 once treated with NSC. The upregulation of TIGAR and SCO2 and co-localization with p53 suggest that NSC may restore the effects of wild-type p53. Current Research / Future Studies: We plan to further characterize whether the alterations in TIGAR and SCO2 expression by NSC are associated with priming EAC cells for therapy. The threshold for apoptosis will be compared between cells treated with NSC versus controls and between cells transduced with short-hairpin RNA targeting TIGAR (shTIGAR) or SCO2 (shSCO2) or both (shTIGAR/SCO2) versus controls. Our hypothesis is that NSC restores the effects of p53 on the mitochondrial via SCO2 and that knockdown of SCO2 will abrogate the effects of NSC. In both NSC treated and short-hairpin transduced cells, we plan to perform standard mitochondrial experiments including JC-1 assay for membrane potential, lactate and ATP production, OCR and ECAR, and ROS generation. Next, we will test for specific alterations in metabolic genes including the apoptotic genes such as Bcl-2, Bcl-XL, Bax, Bak, Bim, Bid, Puma, Noxa, the glycolytic proteins LDHA, GLUT1 and 4, and HK2, and the regulatory proteins such as mTORC1, AMPK, and GSH. p53 mutant protein accumulates at the nucleus69 and localizing to the mitochondria in conjunction with the apoptotic genes Bcl-2 and Bcl-Xl 70,71. Confocal imaging of the mitochondria with counter stains for the nucleus will be used to evaluate whether these drugs or shTIGAR/SCO2 alter mitochondrial shape, size or sub-cellular location.52,61 Next, we will treat shSCO2 and shTIGAR with NSC and control to determine whether knockdown of these proteins blocks the effects of NSC. To date, shSCO2 and shTIGAR are in selection media, but we have not tested the strength of the knockdown. If these lentiviral transductions are inadequate, as a contingency plan we will remove the genes via CRISPR/Cas9 gene editing technique. The goal of this Specific Research Aim is to determine whether p53 inhibition primes EAC cells to increase the vulnerability to metabolic inhibition or standard therapy. Once the experiments listed above determine the effects of NSC on the mitochondrial and whether these effects are abrogated with shSCO2, we plan to directly assess mitochondrial priming through BH3 profiling similar to the description in Project 2. To further evaluate whether p53 mutant inhibition with either mitochondrial inhibition or with cisplatin an in-vivo model will be necessary. We plan to inject Esc3 and Eso26 into the flanks of nude mice. Once the tumors are greater than 200 mm2, the mice would be treated with metabolic inhibitors, p53 mutant inhibitors, cisplatin, and a potential combination of each. The details of this experiment will depend on the above results and have not been specifically planned at this point.